B. Fixation

Collect cells by centrifugation and aspirate supernatant.

Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.

Fix for 10 min at 37°C.

Chill tubes on ice for 1 min.

For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.

Incubate 30 min on ice.

Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

Aliquot 0.5–1 x 106 cells into each assay tube (by volume).

Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.

Specificity / Sensitivity

Src (36D10) Rabbit mAb (Pacific Blue™ Conjugate) detects endogenous levels of Src proteins. The antibody may cross-react with other Src family members, but does not cross-react with other non-family proteins.

Source / Purification

Product Description

This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometry in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody Src (36D10) Rabbit mAb #2109.

Background

The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).

Data Sheets & Documentation

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a trademark of Cell Signaling Technology, Inc.
Pacific Blue is a trademark of Molcular Probes, Inc.
U.S. Patent No. 5,675,063.